This invention relates to a specific set of primers for uniquely determining the presence of bovine Y chromosome. Specifically, it includes unique primers for the amplification by a polymerase chain reaction (PCR) methodology of a specific bovine Y chromosome sequence. This invention also relates to a method for detection of the presence of a bovine male chromosome sequence using specific oligonucleotide primers for amplification of the male chromosome sequence by polymerase chain reaction.
The methodology of polymerase chain reaction (PCR) was invented by Kary Mullis in the mid-1980s and has revolutionized molecular genetics by making possible a whole new approach to the study and analysis of genes. The methodology has become well known since it was invented. PCR allows amplification of a preselected region of DNA and can serve as a highly specific and sensitive detection method (K. B. Mullis and F. A. Faloona, Methods Enzymol. 155:335-350, 1987). Some specific PCR methods have been patented in such patents as U.S. Pat. Nos. 4,683,195 and No. 4,683,202 to Mullis, et al. which are hereby incorporated by reference.
The PCR has also been used for the identification of organisms in complex substrates and to the detection of infectious agents (D. M. Olive, J. Clin. Microbiol. 27: 261-265; B. I. Eisenstein, New Engl. J. Med. 322: 178-183, 1990). Some specific oligonucleotide primers have been developed for detection of pathogenic bacteria by PCR (U.S. Pat. No. 5,494,795). Because of its efficiency and specificity, PCR techniques have been applied extensively to every field in which the molecular techniques are employed and to the detection of presence of a specific gene or a portion of the gene of interest.
Efforts of use of PCR have also been made in the meat and dairy industry. It is practically important to recognize the gender in some applications by detecting the presence of the unique Y chromosome in male cells or tissues. The identification of the presence of any portion of a male chromosome can be important for a number of reasons. One of the reasons is the identification of tissues or meat by gender after the tissues have been removed from the animal and disbursed through marketing channels. By identifying for instance the gender (bull or cow in this case), a determination of proper purchases, representations, and quality of the tissues maybe made. Therefore, efforts of designing appropriate primers have been undertaken and a pair of primers specifically identifying a portion of bovine Y chromsome has been successfully designed by the present inventor. Several aspects of the general bovine sequence had been previously identified prior to the present invention. However, among the aspects unique to this invention are the particular and specific primers complementary to the particular sequence of the bovine male chromosome. Despite attempts by others in the art to test or probe for the Y chromosomes, apparently it escaped those in the field to create such primers uniquely designed to amplify the specific region of the sequence on the Y chromosome to indicate the gender. While other pruners may exist in the marketplace, these primers are unique. By simply using these primers on the tissues, a ready identification of the gender may be made. The prior references do not teach effective or suitable primers to the extent now shown.
Accordingly, an object of this invention is a set of oligonucleotide primers [sequence 5xe2x80x2-GTGATCCGGCATATAGCTGAGA-3xe2x80x2 (SEQ ID No. 1)] and [sequence 5xe2x80x2-TGGTCGCTGATCAGGATGGAA-3xe2x80x2 (SEQ ID No. 2)] for PCR amplification of a portion of bovine Y chromosome DNA sequence.
An additional object of this invention is the demonstration of the specificity of the above oligonucleotide primers for use in detecting the presence of a portion of bovine Y chromosome DNA sequence. This demonstration is specific only to a region of the Y chromosome sequences in bulls but not specific to other chromosome DNA sequences in bulls and chromosome sequences in cows.
These and additional objects of the invention are accomplished by application of standard PCR methodology employing the oligonucleotide primers [5xe2x80x2-GTGATCCGGCATATAGCTGAGA-3xe2x80x2 (SEQ ID No. 1) and 5xe2x80x2-TGGTCGCTGATCAGGATGGAA-3xe2x80x2 (SEQ ID No. 2)] to amplify a portion of the bovine Y chromosome DNA sequence. The bovine species used in this invention in particular is Bos taurus but these primers might be used in other bovine species and, to-some extent, even in species other than bovines.